Quantification and modulation of thrombomodulin activity in isolated rat and human glomeruli

Abstract

Thrombomodulin (TM), the endothelial cell surface receptor for thrombin-mediated activation of protein C and of its anticoagulant system, is involved in maintaining vascular nonthrombogenicity, and depressed TM activity may induce intravascular fibrin formation. TM antigen was previously found by immunohistochemical methods in rabbit glomeruli. We therefore attempted to identify the corresponding TM activity in isolated detergent-solubilized rat and human glomeruli. Like purified lung TM, rat glomeruli extracts accelerated the hydrolysis by activated protein C of the chromogenic substrate S-2238 in the presence of 10 nM thrombin, as determined by spectrophotometry. One mg glomerular protein promoted the formation of 681 +/- 115 nmol activated protein C, the equivalent of the amount generated by 845 ng of purified rabbit TM. TM activity correlated with the protein content of the glomerular extracts (r = 0.94). These extracts prolonged rat plasma activated partial thromboplastin time. Incubation of glomeruli with tumor necrosis factor-alpha (TNF) or E. coli lipopolysaccharide depressed their TM-like activity in a dose and time dependent manner. Incubation with TNF suppressed their anticoagulant activity. In human glomeruli, TM activity was also found at a level which corresponded to their TM antigen content, and was determined by ELISA with mouse monoclonal antibody. These results indicate that measurement of glomerular TM activity might help to clarify the mechanisms of intraglomerular fibrin deposition in renal diseases.

Decrease of thrombomodulin contributes to the procoagulant state of endothelium in hemolytic uremic syndrome

“…Moreover, TM concentration in capillaries is high compared with the concentration seen in large blood vessels. In the kidney, TM is expressed in glomerular EC and might prevent local fibrin formation [3]. The relevance of TM in coagulation processes has been established in mouse models, where mutations in the TM gene lead to its altered cofactor activity and increased levels of fibrin deposits in different organs [4].…”

Puerarin offsets the anticoagulation effect of warfarin in rats by inducing rCyps, upregulating vitamin K epoxide reductase and inhibiting thrombomodulin

“…The VKOR activity was reflected by the amount of vitamin K 1 formed during incubation. The ability of thrombomodulin to accelerate protein C activation was tested using rat lung microsome incubation . The activity of thrombomodulin was assessed by the amount of activated protein C (APC) formed during incubation.…”

“…Approximately 30 mg of rat livers or lungs were homogenized, and RNA was isolated with the Qiagen RNA extraction kit (Valencia, CA, USA). The sequence of the sense and antisense primers for rat VKOR gene (VKORC1) , TM gene , rCyp1a1 , rCyp2b1 , rCyp2c6 , rCyp2c11 and GAPDH gene were designed as described previously. The fold change of target gene expression level from different treatment groups was determined using the equation as follow: Fold change = where and .…”

Radix Puerariae lobatae (Gegen) suppresses the anticoagulation effect of warfarin: a pharmacokinetic and pharmacodynamics study

“…The changes in optical density per minute were converted into units of APC formed by referring to a standard curve constructed with concentrations of 20, 40, 80, 160 and 320 n of standard APC (Stago). The activity of TM could be expressed as the amount of APC formed during incubation [ 47 ].…”